The present invention relates to an apparatus for the separation and fractionation of differentially expressed gene fragments. It relates more particularly to an apparatus and a process for the separation and fractionation of differentially expressed gene fragments (DNA fragments), which are suitable for searching a differentially expressed gene specific to a disease or a function.
Progress in life science and biotechnology has increased the need for the separation and fractionation of the DNA fragments. In particular, in progress in human genome and other genome projects, intensive attempts have been made to analyze the whole of expressed genes in an individual, to extract a differentially expressed gene specific to a certain organ or a gene involved with a specific function or disease, and to analyze the functions of the extracted gene. Accordingly, demands have been made to provide an apparatus which can analyze the whole of a multitude of samples and separate and sample differentially expressed gene (DNA) fragments with efficiency.
Of processes for detecting expression patterns of genes, some processes are based upon the size analysis of DNAs using electrophoresis. Such processes include, for example, the differential display method (hereinafter briefly referred to as xe2x80x9cDD methodxe2x80x9d) (Nucleic Acids Research, Vol. 25, No. 12, pp.2541-2542(1997)), the fluorescence differential display method (hereinafter simply referred to as xe2x80x9cFDD methodxe2x80x9d) (FEBS Lett., 351(2), 231-236, September 1994), the amplified fragments length polymorphism method (hereinafter simply referred to as xe2x80x9cAFLP methodxe2x80x9d) (Nucleic Acids Research, Vol. 23, No. 21, pp. 4407-4414(1995)).
To be more specific, according to these methods, mRNAs are extracted from different biological tissues (e.g., from a normal cell and a cancerous cell), translated to cDNAs through reverse transcription, the resulting cDNAs are fragmented with a restriction enzyme treatment, the resulting fragments are then subjected to amplification by polymerase chain reaction (PCR) using an arbitrary primer (according to the DD method or FDD method) or a selected primer (according to the AFLP method), and the obtained PCR products are electrophoresed for the size separation to compare the obtained electropherograms. By way of illustration, if there is a DNA fragment which is specifically strongly observed in the cancerous cell, the DNA fragment is a candidate for a differentially expressed gene specific to the cancer, and is separated and sampled. According to a conventional procedure, the size separation of DNAs by electrophoresis, and the separation and fractionation of a differentially expressed gene are conducted in the following manner.
By way of illustration, when a slab gel about 0.3 mm thick is used as a separation medium, a mixture (a base length marker) of DNA fragments each having a known length is electrophoresed in some electrophoresis lanes, and a sample DNA to be analyzed, which has been labeled with a fluorophore, is electrophoresed in the other electrophoresis lanes. In general, if base lengths are analyzed using a slab gel, the total amount of the sample DNA is approximately 1 pmol (picomole; 10xe2x88x9212 mole), and the volume of the sample DNA solution is several microliters (xcexcL), each supplied to one electrophoresis lane. The sample is supplied to wells formed at an end of the slab gel, and then a voltage is applied to electrophorese the sample DNA.
After completion of the electrophoresis, an image formed on the gel is read out by an image reader. Such an image reader generally employs a technique of scanning laser light upon the gel surface and imaging the obtained fluorescent intensities to read out the positions of DNA bands in the slab gel.
In the electrophoresis on a slab gel, electrophoresis lanes often bend due to the effect of a distribution of temperature in the gel surface or the like to cause distortions in image. These distortions in image are corrected or calibrated in actual analysis and a base length pattern of the sample DNA is determined by a comparison between the positions of bands of the marker and those of the sample DNA. If a differential band is detected based on the comparison among base length patterns of different samples, the band is cut out from the gel to separate and collect the DNA fragment. The cut-out piece of gel is immersed in a buffer solution, allowed to stand for several hours or overnight to elute the DNA fragment from the gel into the buffer solution. The eluted DNA fragment is purified and then subjected to a sequencing reaction.
The conventional procedure requires a minute and precise technique to cut out a differential band in a precise size from the gel and thus requires a skilled and experienced person to handle. In addition, the extraction and purification of the DNA fragment from the cut-out gel require labor and a long period of time.
The gene expression profiling requires to analyze large amounts of samples, since comparisons between individual organs, between a normal tissue and a disease (e.g., cancer) tissue, or between a parent and a child, for example, are carried out using a combination of several tens of primers. Strong demands have therefore been made to improve working efficiency.
As examples of automatic apparatus for the separation and fractionation of differentially expressed gene fragments (DNA fragments), there may be mentioned one described in Japanese Patent Laid-open No. 7-181164 (hereinafter referred to as xe2x80x9cthe first conventional techniquexe2x80x9d), in which sample DNAs are electrophoresed using capillaries or slab gels each filled with a separation medium, and DNA fragments eluted into sheath flows of a buffer solution, transferred with the flow of the buffer solution by a transfer tube and sampled to sampling vessels. In this apparatus, the vessels are actuated according to detection signals of the DNA fragments eluted into sheath flows to collect a target DNA fragment automatically.
Separately, an apparatus is described in Japanese patent Laid-open No. 6-138037 (hereinafter referred to as xe2x80x9cthe second conventional techniquexe2x80x9d), in which first capillaries and second capillaries are respectively disposed in an optical cell face to face at specified gaps, which optical cell serves to detect DNAs labeled with fluorophores, and the DNA fragments eluted from the first capillaries into the optical cell are transferred to the second capillaries. In this apparatus, inner diameters of the second capillaries are greater than those of the first capillaries to introduce the DNA fragments to the second capillaries with ease and reliability.
Japanese Patent Laid-open No. 7-181164 (the first conventional technique) discloses fundamental constitutive elements of the apparatus, but fails to disclose a practical structure of the transfer tube, a concrete flow rate of the buffer solution and other parameters in a practical manner, which parameters are essential features for determining the performances of separation and fractionation of DNA fragments (in particular precision in separation of the DNA fragments, and required time to collect the DNA fragments). Japanese Patent Laid-open No. 6-138037 (the second conventional technique) lacks descriptions regarding the separation and fractionation of DNA fragments.
Accordingly, it is an object of the invention to improve the apparatus disclosed in Japanese Patent Laid-open No. 7-181164 (the first conventional technique) and thus to provide an apparatus for the separation and fractionation of differentially expressed gene fragments, which can shorten a required time for the separation and fractionation of DNA fragments and provide high separation.
The present inventors reviewed in detail the apparatus (fraction collector) disclosed in Japanese Patent Laid-open No. 7-181164 (the first conventional technique), and found that the performances of the separation and fractionation are significantly affected by a process for transferring DNA fragments through a transfer tube. To be more specific, the flow of a buffer solution in a transfer tube becomes a Poiseuille flow due to the viscosity of the buffer solution, and it is slower in the vicinity of a tube wall than at the center of the transfer tube, and has a flow rate distribution in a cross section perpendicular to the tube axis of transfer tube. As DNA fragments have small diffusion coefficients due to their large molecular weights, they are significantly affected by the distribution in flow rate (differences in flow rate) of buffer solution when they are uniformly injected with respect to the cross section perpendicular to the tube axis of transfer tube, and DNA bands separated by the gel and eluted into the buffer solution are spread out in time. DNA fragments (of base lengths of several tens to hundred) typically have a diffusion coefficient D of 10xe2x88x925 mm2/sec.
Defining an inner diameter of the transfer tube as r and an average flow rate of the buffer solution flowing through the transfer tube as u, a spread xcex94tp in time of DNA fragments in the transfer tube can be determined with reference to the formula (formula (1)) of Goley (edited by Funasaka and Ikegawa, xe2x80x9cContemporary Gas Chromatography (I), Fundamentalsxe2x80x9d pp. 36-46, Hirokawa Shoten, Japan). This formula represents a theoretical plate height, H, as an index of the efficiency of a separation column and is known in the theoretical fields of gas chromatograph and liquid chromatograph.
H=2D/u+(1+6k+11k2)r2u/{24D(1+k)2}xe2x80x83xe2x80x83(1)
where r is an inner diameter of a transfer tube (a capillary), u is an average flow rate of a fluid (a buffer solution) in the transfer tube (capillary) and k is a distribution coefficient.
In the formula of Goley (the formula (1)), when the flow is assumed to be a simple flow in the tube, the distribution coefficient is set to k=0, and the formula 1 is rendered to be the following formula (2). Defining a standard deviation of spatial spread of DNA fragments eluted to the buffer solution in the longitudinal direction of the transfer tube as "sgr" and a length of the transfer tube as L, the parameter H is defined by the following the formula (3):
H=(2D/u+r2u/(24D)xe2x80x83xe2x80x83(2)
H="sgr"2/Lxe2x80x83xe2x80x83(3)
As the diffusion coefficient D of DNA fragments is small, the first term of the formula (2) can be neglected. The spread xcex94tp in time which is calculated according to the formulae (2) and (3) is, therefore, approximated to the following formula (4). To be more specific, when DNA fragments each having a distribution width of xcex4 function are injected to an inlet of the transfer tube in a uniform manner, the width of spatial spread of the DNA fragments in the longitudinal direction of the transfer tube is expressed as "sgr", and xcex94tp is expressed by the formula (4).
xcex94tp="sgr"/u=r{square root over ( )}{L/(24uD)}xe2x80x83xe2x80x83(4)
The results of an experiment with varying L demonstrate that xcex94tp was proportional to {square root over ( )} (L) in the formula (4) and the results of an experiment with varying u demonstrate that xcex94tp was proportional to {square root over ( )} (uxe2x88x921) when DNA fragments were uniformly injected with respect to the cross section perpendicular to the tube axis of transfer tube. Thus, it has been confirmed by experiments that the formula (4) holds well.
The above results demonstrate that the length of transfer tube L is to be shorten, the inner diameter of transfer tube r is to be lessen and the flow rate of buffer solution in the transfer tube is to be increased in order to minimize the spread of bands in the transfer tube. These parameters have, however, limitations in practice. By ascertaining the practical limitations and employing optimum values for the parameters, an apparatus for the separation and fractionation of differentially expressed gene fragments can be provided, which apparatus provides minimized period of time required for the separation and fractionation and improved separation. To reduce the influence of Poiseuille flow, a sampling means for sampling DNA fragments alone which are transferred in the vicinity of the center of the transfer tube or a transferring means of using air flow containing the DNA fragments in the form of droplets can be employed, resulting in minimized spread in time of DNA fragments.
A difference in migration time between the detection of a DNA fragment and that of another adjacent DNA fragment both having different sizes is determined by the length of a capillary as a separation medium, and the migration voltage. By setting the spread in time of bands of DNA fragments caused by a transferring means to be smaller than the difference in migration time of the fragments, a target DNA fragment can be separated and collected.
According to a capillary electrophoresis separator where DNA fragments are separated using a capillary filled with a separation medium, as the capillary has satisfactory heat radiation characteristics, electrophoresis at high voltage can be achieved and hence size analyses at a higher rate can be achieved than an apparatus using a slab gel. By adding a separation and fractionation mechanism to such a capillary electrophoresis separator to make full use of the above characteristics, DNA fragments can be separated and fractionated in a simple manner at a high rate. In contrast to a slab gel, such a capillary requires only trace amount of a prepared sample (in general, approximately 10 nL (nanoliters)) to be injected into the capillary. Separate injection of a sample and a marker into the capillary prevents the prepared sample from contamination. The prepared samples can therefore be used for different multiple applications or objectives, resulting in reduced costs for preparation of samples on the whole of analysis.
Accordingly, the present invention provides, in an aspect, an apparatus for the separation and fractionation of differentially expressed gene fragments, which apparatus includes a separating means including one or a plurality of capillaries each filled with a separation medium to separate DNA fragments by electrophoresis, the DNA fragments each labeled with a fluorophore, a detecting means to apply laser light to the DNA fragments separated by the capillaries and to detect fluorescence emitted from the fluorophore, a transferring means including sampling tubes which are placed with their first ends opposed to the terminal ends of the electrophoresis capillaries at a specified gap, the separated DNA fragments being transferred to the openings of the first ends, a means to form sheath flows of a buffer solution and to carry the separated DNA fragments eluted from each capillary to the openings of the first ends by sheath flows of the buffer solution, a sampling means including sampling vessels to fractionate and sample the DNA fragments according to their sizes, the DNA fragments transferred by the sampling tubes, and a control means to control the sampling means based on a signal gained by the detecting means. The apparatus has the following features:
(1) The sampling tubes each have a length ranging from 5 cm to 15 cm and an inner diameter ranging from 50 xcexcm to 100 xcexcm and the buffer solution flowing through the sampling tubes each have a flow rate of approximately 10 mm/sec.
(2) The apparatus may have a means to form droplets of the buffer solution containing the DNA fragments at the second ends of the sampling tubes, the DNA fragments transferred by the sampling tubes, and a transporting means to transport the formed droplets by airflow to the sampling vessels.
(3) The apparatus may further include a reservoir placed with second ends of the sampling tubes to contain the buffer solution eluted from the second ends, the reservoir having apertures at its bottom, and the apertures each having such a diameter that each sampling tube can be placed therethrough, a first tube to transport a washing fluid to the reservoir, and a second tube to drain the buffer solution from the reservoir, in which the second ends of the sampling tubes penetrate the apertures to transport the DNA fragments transferred by the sampling tubes to the sampling vessels.
(4) The laser light may be applied to the gap or to the capillaries.
(5) The apparatus may include a plurality of the capillaries in the separating means, and a voltage applying means to apply a voltage for electrophoresis to each of the capillaries independently.
(6) The apparatus may have a plurality of the capillaries in the separating means, and a voltage applying means to apply a voltage for electrophoresis to each of the capillaries independently, in which the voltage applying means changes the voltage during electrophoresis based on the signal gained by the detecting means.
(7) The sampling tubes may each have a length ranging from 5 cm to 15 cm and an inner diameter approximately ten times greater than the outer diameter of each capillary, and the buffer solution flowing through the sampling tubes may have a flow rate ranging from 0.3 mm/sec to 1 mm/sec.
The invention provides, in another aspect, an apparatus for the separation and fractionation of differentially expressed gene fragments, which apparatus includes a separating means containing a capillary filled with a separation medium to separate DNA fragments by electrophoresis, the DNA fragments each labeled with a fluorophore, a detecting means to apply laser light to the DNA fragments separated by the capillary and to detect fluorescence emitted from the fluorophore, a first tray to hold wells for holding a sample solution containing the DNA fragments, a second holder to hold wells for holding a marker solution containing a marker, and an injecting means to inject the sample solution and marker solution into the capillary separately.
In a further aspect, the invention provides an apparatus for the separation and fractionation of differentially expressed gene fragments, which apparatus includes a separating means including a capillary filled with a separation medium to separate DNA fragments by electrophoresis, the DNA fragments each labeled with a fluorophore, a detecting means to apply laser light to the DNA fragments separated by the capillary and to detect fluorescence emitted from the fluorophore, a means to form a sheath flow of a buffer solution and to carry the separated DNA fragments eluted from the terminal end of electrophoresis capillary, a sampling means including sampling vessels to fractionate and collect the DNA fragments according to their sizes, a transferring means to transfer the buffer solution containing the DNA fragments to the sampling means, and a control means to control the sampling means based on a signal gained by the detecting means, in which a spread in time of the DNA fragments caused by the transferring means during transfer of the DNA fragments to the sampling vessels is smaller than differences in separation times of the DNA fragments in the separating means, and wherein the transferring means includes a sampling tube which is placed with its end opposed to the terminal end of the electrophoresis capillary at a specified gap, and the separated DNA fragments are transferred to the openings of the first end.
The present invention provides, in yet another aspect, a process for the separation and fractionation of differentially expressed gene fragments, which process includes: a step for injecting a sample DNA solution, and a marker solution into a capillary separately, the sample DNA solution containing DNA fragments each labeled with a fluorophore, the marker solution containing a marker labeled with another fluorophore different from the aforementioned fluorophore with which the DNA fragments are labeled, and the capillary filled with a separation medium, a step for separating the DNA fragments and the marker by electrophoresis, and a step for applying laser light to the DNA fragments and marker separated by the capillary and detecting fluorescence emitted from different species of fluorophores to obtain an electropherogram of the sample DNA.
The invention provides, in a further aspect, a process for the separation and fractionation of differentially expressed gene fragments, which process includes: a step for injecting each of plural sample DNA solutions and a marker solution separately into each of plural capillaries, the DNA solutions containing DNA fragments each labeled with a fluorophore, the marker solution containing a marker labeled with another fluorophore different from the aforementioned fluorophore with which the DNA fragments are labeled, and the capillaries each filled with a separation medium, a step for separating the DNA fragments and the marker by electrophoresis, a step for applying laser light to the DNA fragments and marker separated by each capillary and detecting the fluorescence emitted from different species of fluorophores to obtain electropherograms of the individual sample DNAs and to select a target DNA fragment to be collected based on the electropherograms, a step for separating a sample DNA solution by electrophoresis on a slab gel, the sample DNA solution containing the selected target DNA fragment, and a step for sampling a band of the selected DNA fragment from the slab gel, the DNA fragment separated by electrophoresis.
According to the invention, DNAs of differentially expressed genes can be separated and fractionated automatically with a satisfactory separation, resulting in increased efficiency of researches for pathogenic genes and genes differential to specific functions based upon the gene expression profiling.
An embodiment of the invention can be summarized as follows with reference to FIG. 1: The apparatus includes capillaries 10 to separate DNA fragments each labeled with a fluorophore, detecting means (4a, 6a, 6b, 4b and 2) to apply laser light 28 to the separated DNA fragments and to detect fluorescence emitted therefrom, sampling tubes 14 which are placed with their first ends opposed to the ends of the electrophoresis capillaries at a specified gap, the separated DNA fragments are transferred to the openings of the first ends, means 8 to form sheath flows of a buffer solution and to carry the separated DNA fragments eluted from each of the capillaries to the openings of the first ends by sheath flows of the buffer solution, sampling means 15 including sampling vessels to fractionate and sample the DNA fragments according to their sizes, the DNA fragments being transferred by the sampling tubes, and control means 9, 17 to control the sampling means 15 based on a fluorescence signal. In this embodiment, the sampling tubes each have a length ranging from 5 cm to 15 cm and an inner diameter ranging from 50 xcexcm to 100 xcexcm, and the buffer solution flowing through the sampling tubes each have a flow rate of approximately 10 mm/sec. According to the apparatus of the invention, gene fragments can be separated and fractionated in a short time in a high separation, resulting in increased efficiency of the separation and fractionation procedures of differentially expressed gene fragments.